Culture medium for differentiation of enterobacteriaceae

ABSTRACT

This invention relates to a culture medium for the rapid differentiation and identification of bacteria belonging to the family Enterobacteriaceae.

United States Patent [191- Warren et a1.

[ July 8,1975

[ CULTURE MEDIUM FOR DIFFERENTIATION OF ENTEROBACTERIACEAE [75] Inventors: Bert Warren, Tuxedo Park, N.Y.;

Ronald K. Miller, Fort Lee, NJ.

[73] Assignee: Schering Corporation, Kenilworth,

[22] Filed: Feb. 25, 1974 [21] Appl. No.: 445,507

[56] References Cited UNITED STATES PATENTS 3,832,288 8/1974 Rollender et a1. 195/103.5 R

Primary Examiner-A. Louis Monacell Assistant Examiner-Robert J. Warden Attorney, Agent, or Firm-Arthur E. Wilfond; Bruce M. Eisen; Stephen B. Coan ABSTRACT This invention relates to a culture medium for the rapid differentiation and identification of bacteria belonging to the family Enterobacteriaceae.

{10 Claims, No Drawings (L'LTL'RE MEDIUM FOR DIFFERENTIATION ()F ENTEROBACTERIACEAE This invention relates to culture media for the differentiation and identification of bacteria of the family Enterobacteriaceae. More specifically. it relates to single uniform culture media. and the utilization of such media for the identification ofthe bacteria ofthe family Enterobacteriaceae.

The Enterobacteriaceae are a ubiquitous group of bacteriaconsistingof frank enteric pathogens (Salmonella and Shigella) and many other opportunistic organisms capable of causing infections in every conceivable body locus. They are defined as gram negative rods that reduce nitrates. are oxidase negative and ferment glucose. Their spectrum of sensitivity to antibiotics varies considerably and in many instances therapy ought be based only upon the identity of the organism. Some members are epidemiologically significant and all require identification for specific diagnosis. Curwhich this invention relates have been known and used in routine laboratory identification procedures. such factors have generally employed the use of multiple media. The particular reactions to be described hereinbelow. however. have never been combined in a single uniform multi-reaction medium of this nature. The media of this invention provide a relatively simply prepared single confluent medium. in a butt-slant'configuration. which affords the diagnostician with a number of differential tests in a single tube. The diagnostic test provided by this invention supplements that provided by Warren et al. US. Patent application Ser. No. 357.318 filed May 4. 1973. Other advantages and distinguishing characteristics of this invention over the media of the prior art will also be apparent to the skilled artisan as the details of this invention are explored.

In its broad concept. thisinvention relates to an agarose-base culture medium. and provides identification on the basis of two biochemical reactions; (a) citrate utilization. (b) carbohydrate fermentation. A test utilizing amino acid decarboxylation. is preferably incorporated into the reaction medium.

exemplary citrate substracter are sodium citrate and sodium isocitrate. as well as other citrate salts. Pre

ferred amino acid decarboxylase substrates are ornithine. lysine and arginine and the like. Preferred carbohydrates for fermentation are inositol. rhamnose. and

raffinose.

Citrate media. such as Simmons citrate. have been used in the past to determine if bacteria could grow by utilizing citrate as the sole source of carbon. Positives are clearly visualized by a deep blue slant characteristic of the alkaline aerobic reaction. Addition of carbohydrate had been considered to compromise the utility of this basic test. For example. Christensen citrate sulfide agar. which contains dextrose. in addition to citrate. gives a positive reaction for E. Coli. whereas this organism gives a negative reaction with Simmons citrate.

The outstanding feature of the medium of this invention is that it contains carbohydrate nutrients which give additional information while not compromising the test for citrate utilization.

In summary. the invention provides an agarose-based culture medium for differentiation and identification of bacteria of the family Enterobacteriaceae which comprises pH responsive dye system. a citrate substrate and a carbohydrate substrate selected from inositol. raffinose and rhamnose and preferably an amino acid decarboxylase substrate. The color change produced by the reaction of Enterobacteriaceae bacteria with said carbohydrate substrate on the dye system does not interfere with the color change produced by the reaction of said bacteria with said citrate.

The preferred additional carbon based substrates added to the medium are ornithine hydrochloride and inositol. Although approximated equal amounts of the other above specified reagents may substitute for the ornithine and inositol. the amino acid decarboxylase substrate. e.g. ornithine. although preferred. need not be present in the reaction medium. As the particular amino acid decarboxylase substrate and the addition carbohydrate reactants are used anaerobically in the butt, they do not interfere with the aerobic citrate reaction occuring on the slant. If the organism tested is citrate positive. that is utilizes citrate. ornithine decarboxylation and inositol utilization will occur with all organisms possessing such reactivity. However. if the organism tested cannot use citrate. ornithine and inositol reactivity are not uniformly functional.

Essentially. the preferred medium of this invention is comprised of such ingredients as agar (base medium). brom thymol blue and alizarin red 5 (joint pH indicators). sodium citrate (carbon source). ornithine hydrochloride (detection of ornithine decarboxylase), pyridoxol hydrochloride. p-aminobenzoic acid and thiamine hydrochloride (co-factors and vitamin supplements'in order to trigger primarily the decarboxylase reaction), starch (prolong shelf life). and inositol (additional fermentable substrate and triggers ornithine decarboxylase reaction). Ferrous ammonium sulfate, mono ammonium phosphate, and dibasic potassium phosphate supply nitrogen and mineral requirements. Magnesium chloride enhances the ornithine reaction.

A suitable medium is comprised of the following constituents:

- grams liter Brom thymol blue Preferred within the above medium is that comprised of thefollowing constituents:

grams liter Brom thymol blue 0.05

Alizarin red S 0.016 Sodium citrate 2.0 Ornithine hydrochloride 12.0 Agarose 10.0 Ferrous ammonium sulfate 0.2

Pyridoxol hydrochloride 0.05

-Continued Thiamine hydrochloride 0.001 p-amino benzoic acid 0.001 Starch 1.0 Magnesium chloride 0.33 Monoammonium phosphate 0. l Dihasic potassium phosphate 0.1 lnositol 2.0

About 27.8 grams of the mixture is weighed out. Sufficient distilled water is added to bring the volume to 1,000 ml. The suspension is then heated with constant stirring until the chemicals have gone completely into solution (approximate temp. 100C). The medium is then autoclaved, or heat sterilized at psi for 1 5 minutes. The medium is then dispensed into sterile test tubes sterile 13 X 100 screw cap tubes (4 ml per tube). The agarose may be allowed to cool and harden at an angled position in order to obtain a butt-slant configuration or may be reheated, and cooled and hardened to form a butt-slant configuration just prior to use. The solidified medium prior to use should have a pH of from about 6.0 to about 6.4, preferably 6.2.

The use ofthis medium necessitates primary isolation of pure cultures. A test organism is analyzed by taking a single isolated colony with an inoculating wire, stabbing the butt and streaking the slant. The screw cap is then loosely replaced on the tube. The inoculated culture is placed at 37C and allowed to grow for 24 hours. Results are read 24-48 hours following inoculation.

Biochemical differentiations are based largely upon color and/or growth characteristics.

Specifically. the reaction characteristics are as follows:

Citrate: 1f citrate is utilized, the organism will grow aerobically, that is on the slant. This is evidenced by the appearance of colonies on the slant. Often, but not necessarily always. there may be a rise in pH resulting in a color change from the original color of pale green to light or deep blue. 1f the organism is unable to utilize citrate no growth or color change will occur.

Ornithine: This reaction is an anerobic reaction and hence takes place in the butt. When decarboxylation occurs the pH will increase to 6.6-6.9 indicated by a color change from light or pale green to deep green.

inositol: When inositol is fermented, acid is produced causing the pH indicators to change to a yellow color. This normally only occurs in the butt. However, if there is a concomitant active ornithine decarboxylase enzyme present, the drop in pH due to inositol utilization serves to trigger the decarboxylase enzyme. This reaction will then override the acid produced from fermentation and turn the butt green. in this case, the inositol reaction is not read. When an organism is strongly positive for inositol and negative for ornithine (i.e., Proteus rettgeri) and weakly positive or slow for citrate. the inositol reaction may override the alkaline slant reaction, thus giving an overall yellow color to the medium.

The following table gives some typical reactions of Enterobacteriaceac on the medium of this invention:

Organism Citrate Ornithine inositol Escherichia coli Shigella Edwardsiella Salmonella Arizona Citrobacter Klebsiella -Continued Organism Citrate Ornithine lnositol E. cloacae d E. aerogenes E. hafniae (+1 or Serratia liquefaciens l-. Serratia marcescens I d Proteus ulgaris d Proteus mirabilis or Proteus morgunii d Proteus rettgeri (+1 Providencia' (+1 or -1- d* various. or dependent upon individual species or strains. (+1 may occur slowly in 2-4 days.

We claim:

'1. An agarose-based culture medium for differentiation and identification of bacteria of the family Enterobacteriaceae which comprises a citrate substrate, and as a sole additional carbohydrate substrate, a member selected from the group consisting of inositol, raffinose and rhamnose and a compatable pH responsive dye system, whereby the color change produced by the reaction of Enterobacteriaceae bacteria with said carbohydrate substrate on said dye system does not interfere with the color change produced by the reaction of said bacteria with said citrate.

2. The bacteria culture medium of claim 1, wherein an amino acid decarboxylase substrate is additionally present.

3. The bacteria culture medium of claim 2, wherein said amino acid decarboxylase substrate is selected from the group consisting of ornithine, lysine and arginine.

4; The bacteria culture medium of claim 3, wherein the pH is from about 6.0 to about 6.4.

5. The bacteria culture medium of claim 1, wherein the carbohydrate substrate is inositol and the decarboxylase substrate is ornithine.

6. A culture medium for differentiation and identification of bacteria of the family Enterobacteriaceae which comprises the following ingredients, said ingredients being present in the proportions indicated:

grams/liter B rom thyntol blue 0.04- 0.06 Alizarin red S 0.014 0.018 Sodium citrate 1.8 2.2 Ornithine hydrochloride 10.5 13.5 i Agarosc 7.0 13.0 Ferrous ammonium sulfate 0.1 0.3 Pyridoxol hydrochloride 0.03 0.07 Thiamine hydrochloride 1 0.001 p-amino benzoic acid 0.001 Starch 0.5 1.5 Magnesium chloride 0.23 0.43 Monoammonium phosphate 0.05 0.15 Dibasic potassium phosphate 0.05 0.15 lnositol 1.6 2.4 water q.s. to make 1000 cc.

7. A sterile bacteria culture medium comprising:

grams/liter Brom thymol blue 0.05 Alizarin red S 0.016 Sodium citrate 2.0 Ornithine hydrochloride 12.0 Agarose 10.0 Ferrous ammonium sulfate 0.2 Pyridoxol hydrochloride 0.05 Thiamine hydrochloride 0.001 p-amino benzoic acid 0.001 Starch 1.0 Magnesium chloride 0.33

Continued Monnammonium phosphate 0.] Dibusic potassium phosphate 0. lnusitol 8. A testing vessel containing therein a hardened culture medium. in the proportion. of:

water q.s. to make 1000 cc.

9. A process for differentiation and identification of bacteria of the family Enterobacteriaceae. which comprises inoculating a culture medium having a slant-butt configuration comprising an agarose base. a citrate substrate, a sole additional carbohydrate substrate a member selected from the group consisting of inositol. raffinose and rhamnose allowing the inoculated medium to grow for at about 24 hours at about 37C. followed by identification of bacteria according to the herein described changes for citrate and carbohydrate utiliaztion.

10. The process of claim 9, wherein the culture medium contains an amino acid decarboxylase substrate selected from the group consisting of ornithine. lysine and arginine and the change for amino acid decarboxylase is utilized. 

1. AN AGAROSE-BASED CULTURE MEDIUM FOR DIFFERENTIATION AND IDENTIFICATION OF BACTERIA OF THE FAMILY ENTEROBACTERIACEASE WHICH COMPRISES A CITRATE SUBSTRATE, AND AS A SOLE ADDITIONAL CARBOHYDRATE SUBSTRATE, A MEMBER SELECTED FROM THE GROUP CONSISTING OFF INOSITOL, RAFFINOSE AND RHAMNOSE AND A COMPATABLE PH RESPONSIVE DYE SYSTEM, WHEREBY THE COLOR CHANGE PRODUCED BY THE REACTION OF ENTEROBACTERIACEAE BACTERIA WITH SAID CARBOHYDRATE SUBSTRATE ON SAID DYE SYSTEM DOES NOT INTERFERE WITH THE COLOR CHANGE PRODUCED BY THE REACTION OF SAID BACTERIA WITH SAID CITRATE.
 2. The bacteria culture medium of claim 1, wherein an amino acid decarboxylase substrate is additionally present.
 3. The bacteria culture medium of claim 2, wherein said amino acid decarboxylase substrate is selected from the group consisting of ornithine, lysine and arginine.
 4. The bacteria culture medium of claim 3, wherein the pH is from about 6.0 to about 6.4.
 5. The bacteria culture medium of claim 1, wherein the carbohydrate substrate is inositol and the decarboxylase substrate is ornithine.
 6. A culture medium for differentiation and identification of bacteria of the family Enterobacteriaceae which comprises the following ingredients, said ingredients being present in the proportions indicated:
 7. A sterile bacteria culture medium comprising:
 8. A testing vessel containing therein a hardened culture medium, in the proportion, of:
 9. A process for differentiation and identification of bacteria of the family Enterobacteriaceae, which comprises inoculating a culture medium having a slant-butt configuration comprising an agarose base, a citrate substrate, a sole additional carbohydrate substrate a member selected from the group consisting of inositol, raffinose and rhamnose allowing the inoculated medium to grow for at about 24 hours at about 37*C. followed by identification of bacteria according to the herein described changes for citrate and carbohydrate utiliaztion.
 10. The process of claim 9, wherein the culture medium contains an amino acid decarboxylase substrate selected from the group consisting of ornithine, lysine and arginine and the change for amino acid decarboxylase is utilized. 